Alpha adrenergic receptors are plasma membrane receptors which are located in the peripheral and central nervous systems throughout the body. They are members of a diverse family of structurally related receptors which contain seven putative helical domains and transduce signals by coupling to guanine nucleotide binding proteins (G-proteins).
The alpha adrenergic receptor family of adrenergic receptors (AR) consists of two groups: alpha-1 and alpha-2. Of the alpha-2 group, there are three distinct subtypes denoted alpha-2A, alpha-2B and alpha-2C. The subtypes are derived from different genes, have different structures, unique distributions in the body, and specific pharmacologic properties. (Due to localization of the genes to human chromosomes 10, 2 and 4, the alpha-2A, alpha-2B, and alpha-2C receptors have sometimes been referred to as alpha-2C10, alpha-2C2 and alpha-2C4 receptors, respectively). Like other adrenergic receptors, the alpha-2 receptors are activated by endogenous agonists such as epinephrine (adrenaline) and norepinephrine (noradrenaline), and synthetic agonists, which promote coupling to G-proteins that in turn alter effectors such as enzymes or channels.
The alpha-2 receptors couple to the Gi and Go family of G-proteins. Alpha-2 receptors modulate a number of effector pathways in the cell: inhibition of adenylyl cyclase (decreases cAMP), stimulation of mitogen activated protein (MAP) kinase, stimulation of inositol phosphate accumulation, inhibition of voltage gated calcium channels and opening of potassium channels. (1, 2). The alpha-2 receptors are expressed on many cell-types in multiple organs in the body including those of the central and peripheral nervous systems.
There has been a considerable research effort to clone and sequence the alpha-2AR. For example, the gene encoding the alpha-2A, alpha-2B, alpha-2C subtypes has been cloned and sequenced. (Kobilka et al. Science 238, 650-656 (1987); Regan et al., Lomasney et al. Proc. Nat. Acad. Sci. 87, 5094-5098 (1994)).
Alpha-2BAR
Alpha-2BAR have a distinct pattern of expression within the brain, liver, lung, and kidney, and recent studies using gene knockouts in mice have shown that disruption of this receptor effects mouse viability, blood pressure responses to alpha-2-AR agonists, and the hypertensive response to salt loading. See (13); (14).
It is known that the alpha-2BAR undergoes short-term agonist promoted desensitization (17). This desensitization is due to phosphorylation of the receptor, which evokes a partial uncoupling of the receptor from functional interaction with Gi/Go (18, 19)). Such phosphorylation appears to be due to G protein coupled receptor kinases (GRKs), a family of serine/threonine kinases which phosphorylate the agonist-occupied conformations of many G-protein coupled receptors (20). The phosphorylation process serves to finely regulate receptor function providing for rapid adaptation of the cell to its environment. Desensitization may also limit the therapeutic effectiveness of administered agonists. For the α2BAR, phosphorylation of serines/threonines in the third intracellular loop of the receptor is dependent on the presence of a stretch of acidic residues in the loop that appears to establish the milieu for GRK function (18).
A polymorphism occurring in the gene encoding the alpha-2BAR has been previously reported. This polymorphism has been described as a deletion of three glutamic acid residues in a highly acidic stretch of amino acids in the third intracellular loop of the receptor. (21, 22). However, no pharmacologic studies have been carried out to determine if this polymorphism alters receptor function.
Given the importance of the alpha-2BAR in modulating a variety of physiological functions, there is a need in the art for improved methods to identify polymorphisms and to correlate the identity of these polymorphisms with signaling functions of alpha-2BAR. The present invention addresses these needs and more by providing polynucleotide and amino acid polymorphisms, molecules, and methods for detecting, genotyping and haplotyping the polymorphisms in the alpha-2BAR. The present invention is useful for determining an individual's risk for developing a disease, assist the clinician in diagnosing and prognosing the disease. The present invention also provides methods for selecting appropriate drug treatment based on the identity of such polymorphism.
Alpha-2AAR
Alpha-2AAR are the principal presynaptic inhibitory autoreceptors of central and peripheral sympathetic nerves and inhibit neurotransmitter release in the brain and cardiac sympathetic nerves. (4). Such inhibition of neurotransmitter release in the brain is the basis for the central hypotensive, sedative, anesthetic-sparing, and analgesic responses of alpha-2AAR agonists (5, 6). Indeed, alpha-2AAR agonists such as clonidine and guanabenz are potent antihypertensive agents which act via central presynaptic alpha2AAR (7). The blood pressure and other responses to alpha-2AAR agonists and antagonists, though, are subject to interindividual variation in the human population (7-9). Such variation, of course, can be due to genetic variation in the structure of the receptor itself, its cognate G-proteins, the effectors, or downstream intracellular targets.
Of particular interest are physiologic and genetic studies which suggest that altered alpha-2AAR function can predispose individuals to essential hypertension (7-9). Other physiologic functions of the alpha-2AAR are known. For example, the alpha-2AAR act to inhibit insulin secretion by pancreatic beta-cells, contract vascular smooth muscle, inhibit lipolysis in adipocytes, modulate water and electrolyte flux in renal cells, and aggregate platelets (3). Thus, like what has been shown with beta-AR polymorphisms (12), potential polymorphisms of the alpha-2AAR may act as risk factors for disease, act to modify a given disease, or alter the therapeutic response to agonists or antagonists.
Polymorphisms near the coding regions in the alpha-2A, alpha-2C and dopamine β-hydroxylase (DBH) genes have been reported causing increased levels of norepinephrine in children with attention-deficit hyperactivity disorder (Comings et al. Clin Genet 55, 160-172 (1999)). Indeed, there have been several reports of non-coding region polymorphisms (i.e., in the 5′ and 3′ untranslated region) of the human alpha-2AAR. One report has identified three SNPs in the coding region (Feng et al. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81, 405-410 (1998). In this work, though, no pharmacologic studies were carried out to determine if these polymorphisms alter receptor function.
Given the importance of the alpha-2AAR in modulating a variety of physiological functions, there is a need in the art for improved methods to identify polymorphisms and to correlate the identity of these polymorphisms with signaling functions of alpha-2AAR. The present invention addresses these needs and more by providing nucleic acid and amino acid polymorphisms, molecules, and methods for. identifying the polymorphisms in the alpha-2AAR. The present invention is useful for determining an individual's risk for developing a disease, assist the clinician in diagnosing and prognosing the disease. The present invention also provides methods for selecting appropriate drug treatment based on the identity of such polymorphism.
Alpha-2CAR
Alpha-2CAR plays specific roles in certain central nervous system functions, such as for example, modulation of the acoustic startle reflex, prepulse inhibition, isolation induced aggregation, spatial working memory, development of behavioral despair, body temperature regulation, dopamine and serotonin metabolism, presynaptic control of neurotransmitter release from cardiac sympathetic nerves, central neurons, and postjunctional regulation of vascular tone Sallinen et al. Mol.Pharmacol 51, 36-46 (1997); Sallinen et al. The Journal of Neuroscience 18, 3035-3042 (1998); Tanila et al. European Journal of Neuroscience 11, 599-603 (1999); Bjorklund et al. Mol. Pharmacol 54, 569-576 (1998); Hem et al. Nature 402, 181-184 (1999); Gavin et al. Naunyn Schmiedebergs Arch Pharmacol 355, 406-411(1997); Sallinen et al. Mol Psychiatry 4, 443-452 (1999).
Polymorphisms near the coding regions in the alpha-2A, alpha-2C and dopamine β-hydroxylase (DBH) genes have been reported causing increased levels of norepinephrine in children with attention-deficit hyperactivity disorder (Comings et al. Clin Genet 55, 160-172 (1999).
To date polymorphisms occurring in nucleic acids encoding the alpha-2CAR receptor molecule and in the alpha-2C receptor have not been reported.
Given the importance of the alpha-2CAR in modulating a variety of physiological functions, there is a need in the art for improved methods to identify these polymorphisms and to correlate the identity of these polymorphisms with the physiological functions of alpha-2CAR. The present invention addresses these needs and more by providing nucleic acid and amino acid polymorphisms, molecules, and methods for identifying the polymorphisms in the alpha-2CAR. The present invention is useful for determining an individual's risk for developing a disease and to diagnosis and prognosis the disease. The present invention also provides methods for selecting appropriate drug treatment based on the identity of such polymorphisms.